The primary developmental events of plants originate from the shoot apical meristem (SAM) (Clark, “Organ Formation at the Vegetative Shoot Meristem,” Plant Cell 9:1067-1076 (1997); Kerstetter et al., “Shoot Meristem Formation in Vegetative Development,” Plant Cell 9:1001-1010 (1997)). The shoot apical meristem (SAM) is responsible for the formation of vegetative organs such as leaves, and may undergo a phase change to form the inflorescence or floral meristem. Many of these events are controlled at the molecular level by transcription factors. Transcription factors (TFs) are proteins that act as developmental switches by binding to the DNA (or to other proteins that bind to the DNA) of specific target genes to modulate their expression. An important family of TFs involved in regulating the developmental events in apical meristems is the knox (knotted-like homeobox) gene family (Reiser et al., “Knots in the Family Tree: Evolutionary Relationships and Functions of Knox Homeobox Genes,” Plant Mol Biol 42:151-166 (2000)). Knox genes have been isolated from several plant species (reviewed in Reiser et al., “Knots in the Family Tree: Evolutionary Relationships and Functions of knox Homeobox Genes,” Plant Mol. Biol. 42:151-166 (2000)) and can be divided into two classes based on expression patterns and sequence similarity (Kerstetter et al., “Sequence Analysis and Expression Patters Divide the Maize knotted1-like Homeobox Genes into Two Classes,” Plant Cell 6:1888-1887 (1994)). Class I knox genes have high similarity to the kn1 homeodomain and generally have a meristem-specific mRNA expression pattern. Class II knox genes usually have a more widespread expression pattern.
Knox genes belong to the group of TFs known as the TALE superclass (Bürglin, “Analysis of TALE Superclass Homeobox Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved Between Plants and Animals,” Nucleic Acids Res 25:4173-4180 (1997)). These TFs are distinguished by a very high level of sequence conservation in the DNA-binding region, designated the homeodomain, and consisting of three α-helices similar to the bacterial helix-loop-helix motif (Kerstetter et al., “Sequence Analysis and Expression Patterns Divide the Maize knotted1-like Homeobox Genes into Two Classes,” Plant Cell 6:1877-1887 (1994)). The third helix, the recognition helix, is involved in DNA-binding (Mann et al., “Extra Specificity From extradenticle: the Partnership Between HOX and PBX/EXD Homeodomain Proteins,” Trends in Genet 12:258-262 (1996)). TALE TFs contain a three amino acid loop extension (TALE), proline-tyrosine-proline, between helices I and II in the homeodomain, that has been implicated in protein interactions (Passner et al., “Structure of DNA-Bound Ultrabithorax-Extradenticle Homeodomain Complex,” Nature 397:714-719 (1999)). There are numerous TFs from plants and animals in the TALE superclass and the two main groups in plants are the KNOX and BEL types (Bürglin, “Analysis of TALE Superclass Homeobox Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved Between Plants and Animals,” Nucleic Acids Res 25:4173-4180 (1997)). Related genes in animal systems play an important role in regulating gene expression.
Expression patterns and functional analysis of mutations support the involvement of knox genes in specific developmental processes of the shoot apical meristem. Kn1 from maize, the first plant homeobox gene to be discovered (Vollbrecht et al., “The Developmental Gene Knotted-1 is a Member of a Maize Homeobox Gene Family,” Nature 350:241-243 (1991)), is involved in maintenance of the shoot apical meristem and is implicated in the switch from indeterminate to determinate cell fates (Chan et al., “Homeoboxes in Plant Development,” Biochim Biophys Acta 1442:1-19 (1998); Kerstetter et al., “Loss-of-Function Mutations in the Maize Homeobox Gene, knotted1, are Defective in Shoot Meristem Maintenance,” Development 124:3045-3054 (1997); Clark et al., The CLAVATA and SHOOT MERISTEMLESS Loci Competitively Regulate Meristem Activity in Arabidopsis,” Development 122:1567-1575 (1996)). Transcripts of kn1 in maize (Jackson et al., “Expression of Maize KNOTTED1 Related Homeobox Genes in the Shoot Apical Meristem Predicts Patterns of Morphogenesis in the Vegetative Shoot,” Development 120:405-413 (1994)), OSH1 in rice (Sentoku et al., “Regional Expression of the Rice KN1-type Homeobox Gene Family During Embryo, Shoot, and Flower Development,” Plant Cell 11: 1651-1663 (1999)), and NTH15 in tobacco (Tamaoki et al., “Ectopic Expression of a Tobacco Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology and Hormone Levels in Transgenic Tobacco,” Plant Cell Physiol 38:917-927 (1997)) were localized by in situ hybridization to undifferentiated cells of the corpus and the developing stem, but were not detected in the tunica or leaf primordia. Overexpression of kn1 in Arabidopsis (Lincoln et al., “A knotted1-like Homeobox Gene in Arabidopsis is Expressed in the Vegetative Meristem and Dramatically Alters Leaf Morphology When Overexpressed in Transgenic Plants,” Plant Cell 6:1859-1876 (1994)) and in tobacco (Sinha et al., “Overexpression of the Maize Homeobox Gene, KNOTTED-1, Causes a Switch From Determinate to Indeterminate Cell Fates,” Genes Dev 7:787-795 (1993)), resulted in plants with altered leaf morphologies including lobed, wrinkled or curved leaves with shortened petioles and decreased elongation of veins. Plants were reduced in size and showed a loss of apical dominance. In plants with a severe phenotype, ectopic meristems formed near the veins of leaves indicating a reversion of cell fate back to the indeterminate state (Sinha et al., “Overexpression of the Maize Homeobox Gene, KNOTTED-1, Causes a Switch From Determinate to Indeterminate Cell Fates,” Genes Dev 7:787-795 (1993)). Overexpression of OSH1 or NTH15 in tobacco resulted in altered morphologies similar to the 35S-kn1 phenotype (Sato et al., “Abnormal Cell Divisions in Leaf Primordia Caused by the Expression of the Rice Homeobox Gene OSH1 Lead to Altered Morphology of Leaves in Transgenic Tobacco,” Mol Gen Genet 251:13-22 (1996); Tamaoki et al., “Ectopic Expression of a Tobacco Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology and Hormone Levels in Transgenic Tobacco,” Plant Cell Physiol 38:917-927 (1997)).
Alterations in leaf and flower morphology in 35S-NTH15 or OSH1 transgenic tobacco were accompanied by changes in hormone levels. Whereas levels of all the hormones measured were changed slightly, both gibberellin and cytokinin levels were dramatically altered (Kusaba et al., “Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1,” Plant Physiol 116:471-476 (1998); Tamaoki et al., “Ectopic Expression of a Tobacco Homeobox Gene, NTH15, Dramatically Alters Leaf Morphology and Hormone Levels in Transgenic Tobacco,” Plant Cell Physiol 38:917-927 (1997)). RNA blot analysis revealed that the accumulation of GA 20-oxidase1 mRNA was reduced several fold in transgenic plants (Kusaba et al., “Decreased GA1 Content Caused by the Overexpression of OSH1 is Accompanied by Suppression of GA 20-oxidase Gene Expression,” Plant Physiol 117:1179-1184 (1998); Tanaka-Ueguchi et al., “Overexpression of a Tobacco Homeobox Gene, NTH15, Decreases the Expression of a Gibberellin Biosynthetic Gene Encoding GA 20-oxidase,” Plant J 15:391-400 (1998)). A KNOX protein of tobacco binds to specific elements in regulatory regions of the GA 20-oxidase1 gene of tobacco to repress its activity (Sakamoto et al., KNOX Homeodomain Protein Directly Suppresses the Expression of a Gibberellin Biosynthesis Gene in the Tobacco Shoot Apical Meristem,” Genes Dev 15:581-590 (2001)). GA 20-oxidase is a key enzyme in the GA biosynthetic pathway necessary for the production of the physiologically inactive GA20 precursor of active GA1 (Hedden et al., “Gibberellin Biosynthesis: Enzymes, Genes and Their Regulation,” Annu Rev Plant Physiol Plant Mol Biol 48:431-460 (1997)). GA1 and other active GA isoforms are important regulators of stem elongation, the orientation of cell division, the inhibition of tuberization, flowering time, and fruit development (Jackson et al., “Control of Tuberisation in Potato by Gibberellins and Phytochrome,” B. Physiol Plant 98:407-412 (1996); Hedden et al., “Gibberellin Biosynthesis: Enzymes, Genes and Their Regulation,” Annu Rev Plant Physiol Plant Mol Biol 48:431-460 (1997); Rebers et al., “Regulation of Gibberellin Biosynthesis Genes During Flower and Early Fruit Development of Tomato,” Plant J 17:241-250 (1999)).
Another plant homeobox gene family that is closely related to the knox genes is the BEL (BELL) family (Chan et al., “Homeoboxes in Plant Development,” Biochim Biophys Acta 1442:1-19 (1998); Bürglin, “Analysis of TALE Superclass Homeobox Genes (MEIS, PBC, KNOX, Iroquois, TGIF) Reveals a Novel Domain Conserved Between Plants and Animals,” Nucleic Acids Res 25:4173-4180 (1997)). BEL TFs have been implicated in flower and fruit development (Reiser et al., The BELL1 Gene Encodes a Homeodomain Protein Involved in Pattern Formation in the Arabidopsis Ovule Primordium,” Cell 83:735-742 (1995); Dong et al., “MDH1: an Apple Homeobox Gene Belonging to the BEL1 Family,” Plant Mol Biol 42:623-633 (2000)). Genetic analysis of BEL1 in Arabidopsis showed that expression of this TF regulated the development of ovule integuments and overlaps the expression of AGAMOUS (Ray et al., “Arabidopsis Floral Homeotic Gene BELL (BEL1) Controls Ovule Development Through Negative Regulation of AGAMOUS Gene (AG),” Proc Natl Acad Sci USA 91:5761-5765 (1994); Reiser et al., The BELL1 Gene Encodes a Homeodomain Protein Involved in Pattern Formation in the Arabidopsis Ovule Primordium,” Cell 83:735-742 (1995); Western et al., “BELL1 and AGAMOUS Genes Promote Ovule Identity in Arabidopsis thaliana,” Plant J 18:329-336 (1999)). In COP1 mutants, the photoinduced expression of ATH1, another BEL TF of Arabidopsis, was elevated, indicating a possible role in the signal transduction pathway downstream of COP1 (Quaedvlieg et al., “The Homeobox Gene ATH1 of Arabidopsis is Depressed in the Photomorphogenic Mutants cop1 and det1,” Plant Cell 7:117-129 (1995)).
Plants must maintain a great deal of flexibility during development to respond to environmental and developmental cues. Responses to these signals, which include day length, light quality or quantity, temperature, nutrient and hormone levels, are coordinated within the meristem (Kerstetter et al., “Shoot Meristem Formation in Vegatative Development,” Plant Cell 9:1001-1010 (1997)). In potato, there is a specialized vegetative meristem called the stolon meristem that develops as a horizontal stem and under inductive conditions will form the potato tuber (Jackson, “Multiple Signaling Pathways Control Tuber Induction in Potato,” Plant Physiol. 119:1-8 (1999); Fernie et al., “Molecular and Biochemical Triggers of Potato Tuber Development,” Plant Physiol. 127:1459-1465 (2001)). Potato offers an excellent model system for examining how vegetative meristems respond to external and internal factors to control development at the molecular level. In model tuberization systems, synchronous tuber formation occurs under inductive conditions and shoot or stolon formation occurs under noninductive conditions. The cellular and biochemical processes that occur in these model systems have been examined extensively (Vreugdenhil et al., “Initial Anatomical Changes Associated with Tuber Formation on Single-Node Potato (Solanum tuberosum L.) Cuttings: A Re-evaluation,” Ann. Bot. 84:675-680 (1999); Xu et al., “The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation In vitro,” Plant Physiol. 117:575-584 (1998); Hannapel, “Characterization of Early Events of Potato Tuber Development,” Physiol. Plant 83:568-573 (1991); Wheeler et al., “Comparison of Axillary Bud Growth and Patatin Accumulation in Potato Leaf Cuttings as Assays for Tuber Induction,” Ann. Bot. 62:25-30 (1988)). In addition to being good systems to examine integration of signals at the meristem, understanding the molecular processes controlling tuberization in potato is important. Potato is the fourth largest crop produced in the world, ranking after maize, rice, and wheat, and is a major nutritional source in many countries (Jackson, “Multiple Signaling Pathways Control Tuber Induction in Potato,” Plant Physiol. 119:1-8 (1999); Fernie et al., “Molecular and Biochemical Triggers of Potato Tuber Development,” Plant Physiol. 127:1459-1465 (2001)); therefore, research focusing on the process of tuber initiation and development is very important.
Tuber formation in potatoes (Solanum tuberosum L.) is a complex developmental process that requires the interaction of environmental, biochemical, and genetic factors. Several important biological processes like carbon partitioning, signal transduction, and meristem determination are involved (Ewing et al., “Tuber Formation in Potato: Induction, Initiation and Growth,” Hort. Rev. 14:89-198 (1992)). Under conditions of a short-day photoperiod and cool temperature, a transmissible signal is activated that initiates cell division and expansion and a change in the orientation of cell growth in the subapical region of the stolon tip (Ewing et al., “Tuber Formation in Potato: Induction, Initiation and Growth,” Hort. Rev. 14:89-198 (1992); Xu et al., “Cell Division and Cell Enlargement During Potato Tuber Formation,” J. Expt. Bot. 49:573-582 (1998)). In this signal transduction pathway, perception of the appropriate environmental cues occurs in leaves and is mediated by phytochrome and gibberellins (van den Berg et al., “Morphology and (14C) gibberellin A-12 Metabolism in Wild-Type and Dwarf Solanum tuberosum ssp. Andigena Grown Under Long and Short Photoperiods,” J. Plant Physiol. 146:467-473 (1995); Jackson et al., “Phytochrome B Mediates the Photoperiodic Control of Tuber Formation in Potato,” Plant J. 9:159-166 (1996); Jackson et al., “Control of Tuberisation in Potato by Gibberellins and Phytochrome,” B. Physiol Plant 98:407-412 (1996)). Tuber development at the stolon tip is comprised of biochemical and morphological processes. Both are controlled by differential gene expression (Hannapel, “Characterization of Early Events of Potato Tuber Development,” Physiol. Plant 83:568-573 (1991); Bachem et al., “Analysis of Gene Expression During Potato Tuber Development,” Plant J. 9:745-753 (1996); Macleod et al., “Characterisation of Genes Isolated from a Potato Swelling Stolon cDNA Library,” Pot. Res. 42:31-42 (1999)) with most of the work focusing on the biochemical processes, including starch synthesis (Abel et al., “Cloning and Functional Analysis of a cDNA Encoding a Novel 139 kDa Starch Synthase from Potato (Solanum tuberosum L.),” Plant J. 10:981-991 (1996); Preiss, “ADPglucose Pyrophosphorylase: Basic Science and Applications in Biotechnology,” Biotech. Annu. Rev. 2:259-279 (1996); Geigenberger et al., “Overexpression of Pyrophosphatase Leads to Increased Sucrose Degradation and Starch Synthesis, Increased Activities of Enzymes for Sucrose-Starch Interconversions, and Increased Levels of Nucleotides in Growing Potato Tubers,” Planta 205:428-437 (1998)) and storage protein accumulation (Mignery et al., “Isolation and Sequence Analysis of cDNAs for the Major Potato Tuber Protein, Patatin,” Nucl. Acid Res. 12:7989-8000 (1984); Hendriks et al., “Patatin and Four serine Protease Inhibitor Genes are Differentially Expressed During Potato Tuber Development,” Plant Mol. Biol. 17:385-394 (1991); Suh et al., “Proteinase-Inhibitor Activity and Wound-Inducible Expression of the 22-kDa Potato-Tuber Proteins,” Planta 184:423-430 (1991)).
Much less is known about the morphological controls of tuberization, although it is clear that phytohormones play a prominent role (Koda et al., “Potato Tuber-Inducing Activities of Jasmonic Acid and Related Compounds,” Phytochemistry 30:1435-1438 (1991); Xu et al., “The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation In vitro,” Plant Physiol. 117:575-584 (1998), Sergeeva et al., “Tuber Morphology and Starch Accumulation are Independent Phenomena: Evidence from ipt-transgenic Potato Lines,” Physiol. Plant 108:435-443 (2000)). Gibberellins (GA), in particular, play an important role in regulating tuber development. High levels of GA are correlated with the inhibition of tuberization, whereas low levels are associated with the induction of tuber formation (Jackson et al., “Control of Tuberisation in Potato by Gibberellins and Phytochrome,” B. Physiol Plant 98:407-412 (1996); Xu et al., “The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation In vitro,” Plant Physiol. 117:575-584 (1998)). Specific genes, such as lipoxygenases (Kolomiets et al., “Lipoxygenase is Involved in the Control of Potato Tuber Development,” Plant Cell 13:613-626 (2001)) and MADS box genes (Kang et al., “Nucleotide Sequences of Novel Potato MADS-box cDNAs and their Expression in vegetative Organs,” Gene 166:329-330 (1995)) that are involved in regulating tuber formation have been identified.
Three independent research groups have recently confirmed that BEL-like TFs interact via protein binding with their respective knox-types in three separate species (Bellaoui et al., “The Arabidopsis BELL1 and KNOX TALE Homeodomain Proteins Interact Through a Domain Conserved Between Plants and Animals,” Plant Cell 13:2455-2470 (2001); Müller et al., “In vitro Interactions Between Barley TALE Homeodomain Proteins Suggest a Role for Protein-Protein Associations in the Regulation of Knox Gene Function,” Plant J. 27:13-23 (2001); Smith et al., “Selective Interaction of Plant Homeodomain Proteins Mediates High DNA-Binding Affinity,” Proc. Nat'l. Acad. Sci. USA 99:9579-9584 (2002)), but to date, there is no published report on the function of this interaction. Moreover, nothing is known about the role of either KNOX or the BEL TFs in the regulation of development of tuberous plants, such as potato.
The present invention is directed to overcoming these and other deficiencies in the art.